利用脱细胞猪角膜基质膜片构建组织工程化兔角膜基质

发布时间：2018-10-12 10:56

【摘要】：目的探讨氯化钠法及冰冻切片法制备脱细胞猪角膜基质膜片的可行性,并进一步复合兔角膜基质细胞构建组织工程角膜基质,探讨脱细胞角膜基质膜片在兔角膜基质缺损中的修复效果,为角膜基质的组织工程修复治疗提供实验依据。方法将去除上皮层和内皮层的全层猪角膜基质瓣采用氯化钠法脱细胞,组织学检测脱细胞效率,冰冻切片法将脱净细胞的厚基质瓣切成20μm厚度的脱细胞角膜基质膜片,紫外线照射灭菌保存。采用组织块法分离培养兔角膜基质细胞,传至第三代作为构建组织工程角膜基质的种子细胞。将培养的兔角膜基质细胞以三明治法接种于脱细胞角膜基质膜片支架,层层叠加6层膜片形成支架-细胞复合物,体外培养2周后组织学检测组织形成状态。构建兔角膜基质缺损模型,将膜片和基质细胞按照三明治法移植至角膜基质缺损部位,共叠加5层膜片,另设单纯缺损不修复组、厚基质移植组(100μm)、单纯膜片无细胞组(5层)为对照组,术后第1月,3月和6月进行大体拍照观察、眼前节光学相干断层扫描、超声角膜测厚、角膜透光度检测、生物力学检测、组织学检查、透射电镜超微结构观察等,评价治疗效果。结果氯化钠法能有效脱除猪角膜基质中的角膜基质细胞,同时能良好地维持基质胶原纤维的形态和排列,在脱细胞基质的基础上制作脱细胞基质膜片简单易行。组织块法培养的兔角膜基质细胞体外生长良好,细胞接种至脱细胞猪角膜基质膜片支架材料上增殖良好,扫描电镜检测显示细胞在膜片上贴附生长良好。体外构建组织分层明显,细胞存活但基质分泌少。兔角膜基质移植修复实验中,单纯缺损不修复组角膜菲薄,局部白色瘢痕及新生血管长入;厚基质移植组透明度尚可,瞳孔区可见云翳,随时间的延长云翳略减淡;单纯膜片无细胞组无明显瘢痕,但可见少量膜片皱褶,角膜欠透明,呈斑翳形态;脱细胞基质膜片接种角膜细胞组3个月时可见缝线周围新生血管生成,6个月时逐渐减退,角膜透明。眼前节光学相干断层扫描显示单纯缺损组基质信号不均,有高信号区的疤痕组织;厚膜片组可见移植膜片与原基质间有明显的界限,融合欠佳;单纯膜片组角膜基质层信号欠均匀,可见裂隙状低信号区,表明膜片间粘合欠佳;膜片加细胞组基质信号较均匀,移植膜片间融合良好。厚度检测发现单纯缺损组最薄,其次为单纯脱细胞基质膜片移植组,脱细胞基质移植组和膜片加细胞移植组的厚度均与正常组接近。脱细胞基质膜片加角膜细胞移植组在各波长可见光下的透光率与正常角膜最为接近。与正常对照组相比,单纯缺损组的生物力学检测值均最低,而脱细胞膜片加细胞移植组术后6个月的最大荷载,抗拉强度和弹性模量均与正常组十分接近。组织学检查发现单纯缺损组的角膜厚度显著变薄,虽然厚基质移植组,及单纯基质膜片移植组的厚度有所增加,但未见细胞分布,而膜片加细胞移植组见细胞均匀分布。透射电镜检测中,膜片加细胞移植组的胶原纤维直径与正常组无统计学意义的差异。结论氯化钠法加冰冻切片法制作脱细胞猪角膜基质膜片简单易行。脱细胞猪角膜基质膜片用组织块法培养的兔角膜基质细胞在脱细胞猪角膜基质膜片支架上生长良好,采用膜片与接种细胞叠加的方法能使角膜基质细胞较均匀地分布在基质层中,利于新生角膜基质的生成和修复缺损组织。为构建组织工程角膜基质提供新思路。
[Abstract]:Objective To study the feasibility of preparing acellular porcine corneal stroma membrane by sodium chloride method and frozen section method, and further compound rabbit corneal stroma cells to construct tissue engineering corneal stroma, to investigate the effect of acellular corneal stroma membrane on rabbit corneal stroma defect. provides experimental basis for tissue engineering restoration treatment of corneal stroma. Methods The whole-layer porcine corneal stroma flap was removed by sodium chloride method and the decellular efficiency was examined histologically by using sodium chloride method. The acellular corneal stroma membrane with thickness of 20. m u.m was cut by freeze-section method and sterilized and preserved by ultraviolet irradiation. The rabbit corneal stromal cells were isolated and cultured by tissue mass spectrometry, and transferred to the third generation as seed cells for constructing tissue engineering corneal stroma. The cultured rabbit corneal stroma cells were inoculated on the acellular corneal stroma membrane scaffold by sandwich method, and 6 layers of bone were superimposed on each layer to form the scaffold-cell complex, and the tissue formation was detected after 2 weeks culture in vitro. The rabbit corneal stroma defect model was constructed, the membrane and stromal cells were transplanted to the corneal stroma defect site according to the sandwich method, 5 layers of membrane were superimposed, the other was no repair group, the thick matrix transplantation group (100. mu.m), the simple membrane cell-free group (5 layers) was the control group. In the first month, March and June after operation, general photo observation, anterior segment optical coherence tomography, ultrasonic corneal thickness measurement, corneal topography examination, biomechanical testing, histological examination, transmission electron microscope ultrastructural observation, etc. were performed to evaluate the therapeutic effect. Results Sodium chloride can effectively remove corneal stroma cells in porcine corneal stroma, and can maintain the morphology and arrangement of matrix collagen fibers well, and make acellular matrix membrane simple and feasible on the basis of acellular matrix. The rabbit corneal stromal cells cultured in the tissue mass method were well-grown in vitro, and the cells were inoculated to the acellular porcine corneal stroma membrane scaffold for good proliferation. Scanning electron microscopy (SEM) showed that the cells were well-grown on the membrane. Tissue stratification was evident in vitro, but cell survival was less than that of stroma. In the rabbit corneal stroma transplantation and repair experiment, the pure defect did not repair the corneal firthin, the local white blood line and the newborn blood vessel length; the transparency of the thick matrix transplantation group was fair, the pupil area could be seen to be cloudy, and the cloud length was slightly decreased over time; the pure membrane cell-free group had no obvious effect. However, a small number of membrane wrinkles were found, the cornea was undertransparent and in the form of speckles, and when the acellular matrix membrane was inoculated with corneal cell group for 3 months, the formation of new blood vessels around the suture was seen, and the cornea was gradually decreased in 6 months, and the cornea was transparent. The anterior segment optical coherence tomography showed that the matrix signal of the simple defect group was uneven and the scar tissue in the high signal region was found; the thick film group showed obvious boundary between the graft membrane and the original substrate; the fusion was poor; the corneal stroma layer signal of the simple membrane group was under-uniform and the slit-shaped low signal region was visible. It is shown that the adhesion between the membranes is poor; the matrix signal of the membrane plus cell group is more uniform, and the fusion between the transplanted membranes is good. The thickness test showed that the pure defect group was the thinnest, and then the thickness of the acellular matrix membrane transplantation group, the acellular matrix transplantation group and the membrane plus cell transplantation group were close to that of the normal group. The light transmittance of the acellular matrix membrane plus corneal cell transplantation group at each wavelength of visible light was the closest to that of the normal cornea. Compared with the normal control group, the biomechanical detection value of the pure defect group was the lowest, while the maximum load, tensile strength and elastic modulus were very close to the normal group after 6 months after the cell membrane plus cell transplantation group. Histologic examination revealed a significant thinning of the corneal thickness in the pure defect group, although the thickness of the thick matrix graft group and the simple matrix membrane graft group increased, but no cell distribution was found, whereas the membrane plus cell transplantation group saw a uniform distribution of cells. There was no statistically significant difference between the diameter of collagen fibers in membrane plus cell transplantation group and normal group during transmission electron microscope (TEM). Conclusion The preparation of acellular porcine corneal stroma membrane by using sodium chloride method and frozen section method is simple and easy. The rabbit corneal stroma cells cultured by the tissue mass method of the acellular porcine corneal stroma membrane are well grown on the acellular porcine corneal stroma membrane support, and the method for stacking the membrane and the inoculated cells enables the corneal stroma cells to be uniformly distributed in the matrix layer, is beneficial to the generation and repair of the new corneal stroma and repair the defect tissue. and provides a new idea for constructing tissue engineering corneal stroma.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R772.2

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